Gene Regulation by Antisense
A principal scheme by which information is transferred from a gene is, first, by transcription of the gene to form a corresponding RNA message ("messenger RNA" or "mRNA"), then by translation of the mRNA to form a corresponding protein. This seemingly simple scheme is effected by complex mechanisms in living cells. This scheme is further complicated by the fact that not all genes in a cell's genome are expressed at the same time. Cells possess complex gene regulatory mechanisms that selectively turn genes on and off, thereby determining cell structure, function, and developmental fate.
Certain classes of genes in cells are termed "regulatory genes" because they regulate the expression of other genes. One class is comprised of regulatory genes encoding regulator proteins that interact with other specific genes at the transcriptional or translational level to modulate the amount of protein produced by the regulated gene, often in response to environmental cues. Other classes of regulatory genes function by means other than by producing a regulatory protein.
Mounting evidence indicates that an important naturally occurring means of gene regulation is by "antisense" transcripts, particularly in procaryotic cells. Tomizawa et al., Proc. Natl. Acad. Sci. USA 78:1421-1425 (1981); Mizuno et al., Proc. Natl. Acad. Sci. USA 81:1966-1970 (1984); Simons, Gene 72:35-44 (1988). (Although RNAs from eucaryotic cells have been found that are complementary to known genes, an in vivo regulatory role of these RNAs has not been proven. Van der Krol et al., Biotechniques 6:958-976 (1988)). An antisense transcript, termed "antisense RNA", is comprised of a nucleotide sequence complementary to either an mRNA encoded by a regulated gene or to the "sense strand" of DNA comprising the regulated gene. One probable mechanism by which an antisense RNA regulates a gene is by the formation of a hybrid RNA duplex of the antisense RNA with a particular mRNA via Watson-Crick base pairing. Van der Krol et al., Gene 72:45-50 (1988). Such hybrids appear to be resistant to translation for any of a number of reasons including unusually rapid degradation of the duplex in the cell, impairment of post-transcriptional processing, or inhibition of ribosome binding. Simons, Gene 72:35-44 (1988 ); Van der Krol et al., Gene 72:45-50 (1988). Genes that encode antisense RNAs are termed "antisense genes".
After the discovery of antisense RNAs, researchers began investigating ways to artificially regulate gene expression using antisense RNAs. These studies have proven effective in identifying specific genes, characterizing gene function, controlling infections, and manipulating metabolic pathways. Van der Krol et al., BioTechniques 6:958-976 (1988). For example, antisense RNA synthesized in vitro and introduced into eucaryotic cells can regulate expression of specific genes within the cells, at least temporarily. Izant and Weintraub, Cell 36:1007-1015 (1984); Izant and Weintraub, Science 229:345-352 (1985); Van der Krol et al., BioTechniques 6:958-976 (1988). These experiments have not only been performed using mammalian cells but also plant cells. Van der Krol et al., Gene 72:45-50 (1988); Smith et al., Nature 334:724-726 (1988); Sheehy et al., Proc. Natl. Acad. Sci, USA 85:8805-8809 (1988); Van der Krol et al., Nature 333:866-869 (1988); Ecker et al., Proc. Natl. Acad. Sci. USA 83:5372-5376 (1986); and Rothstein et al., Proc, Natl. Acad. Sci. USA 84:8439-8443 (1987). simultaneous development of gene sequencing, gene cloning, and in vitro gene and RNA synthesis technology made it possible to readily generate antisense RNAs to target genes, provided the target gene or the nucleotide sequence of the target gene was available. Inouye, Gene 72:25-34 (1988).
Antisense RNA can be used to mimic mutations in both procaryotic and eucaryotic organisms. Takayama and Inouye, Crit. Rev. in Biochem. and Molec, Biol., CRC Press 25:155-184 (1990); van der Krol et al., BioTechniques 958-976 (1987). In plants, antisense RNA has been successfully used to inhibit the activity of nopaline synthase, Rothstein et al., Proc. Natl. Acad. Sci. USA 84:8439-8443 (1987); Sandler et al., Plant Mol. Biol. 11:301-310 (1988); chloramphenicol acetyltransferase, Ecker and Davis, Proc. Natl. Acad. Sci, USA 83:5372-5376 (1986); Delauney et al., Proc. Natl. Acad. Sci. USA 85:4300-4304 (1900); chalcone synthase, van der Krol et al., Nature 333:866-869 (1988); polygalacturonase, Smith et al., Nature 334:724-726 (1988); Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); .beta.-glucuronidase, Robert et al., Plant Mol. Biol. 13:399-409 (1989); and granule-bound starch synthase, Visser et al., Mol. Gen. Genet. 225:289-296 (1991).
Because mRNAs have only a limited life in cells, antisense RNA injected into cells generally exhibits a regulatory function of relatively short duration. However, functional antisense genes can be made in vitro by coupling a functional promoter to a DNA sequence oriented in a way wherein the promoter induces transcription of the noncoding (or "nonsense") strand of the DNA, rather than the normal coding (or "sense") strand. One approach is to invert the coding sequence of the DNA relative to the promoter. Izant and Weintraub, Science 229:345-352 (1985). This can be done by excising the coding region of the gene, proximal to the promoter and polyadenylation sites, and reinserting the excised portion in reverse orientation relative to the promoter. Izant and Weintraub, Cell 36:1007-1015 (1984). Thus, the antisense gene is transcribed in a direction opposite to the direction of transcription of the corresponding sense gene. Mizuno et al., Proc. Natl Acad. Sci USA 81:1966-1970 (1984). When such an antisense gene is introduced into a recipient cell having an endogenous corresponding "sense" gene, the promoter directs transcription of the nonsense DNA strand, producing a transcript (antisense RNA) complementary to mRNAs normally produced by the corresponding "sense" gene. Introduction of an antisense gene into a cell has been found to result in a more prolonged, or stable, regulatory effect than introduction of antisense RNA, so long as the introduced antisense gene continues to be transcribed by the cell. Of course, integrating the introduced antisense gene into the cell genome can result in continued expression of the antisense gene over the lifetime of the cell.
Many plant cells, in contrast to the vast majority of other eucaryotic cells, are totipotent. That is, individual totipotent plant cells can be readily induced to divide and form entire plants. Hence, introduction of an exogenous antisense regulatory gene into a totipotent plant cell, wherein the antisense gene integrates into the cellular genome, can yield a source of "genetically engineered" plants expressing the new antisense gene in their cells and passing the antisense gene to their progeny. Rothstein et al., Proc. Natl. Acad. Sci. USA 84:8439-8443 (1987).